RLC-D,D–polarized cells create a region containing large adhesions with low Tyr phosphorylation at the rear and small, highly Tyr-phosphorylated, p130(Cas)-containing dynamic adhesions at the front. (A) CHO.K1 cells were transfected with wild-type RLC or RLC-D,D (fused to GFP), stained for phosphorylated Tyr118 paxillin and endogenous vinculin, and imaged with a confocal microscope (FV1000; Olympus). Arrowheads point to several clusters of pY(118)-paxillin at the leading edge; the arrow points to larger accumulations of vinculin adjacent to the RLC-D,D–decorated bundles. Representative morphologies are shown. (B) Quantification of the phosphorylation ratio using the endogenous staining pairs indicated. Data are the mean ± SD (error bars) of the ratio of the relative intensities at the front and the back (back and sides in wild-type cells). 12 cells/condition (adhesion, n > 50) were analyzed from three independent experiments. P represents significance using a Student’s t test. (C) CHO.K1 cells were transfected with wild-type RLC (top) or RLC-D,D (fused to mCherry, bottom) and GFP-p130(Cas), stained for phosphorylated Tyr(165)-p130(Cas), and imaged as in A. (D) TIRF microscopy time-lapse series of a CHO.K1 cell transfected with mGFP-dSH2 and RLC-D,D–mCherry and allowed to spread on fibronectin. Images were collected starting 5 min after plating. Video 7 shows the entire sequence. (E) TIRF microscopy time-lapse series of a CHO.K1 cell transfected with mGFP-dSH2 and RLC-D,D–mCherry migrating on fibronectin. The cell was allowed to spread and polarize for 35 min before image collection began. Video 8 shows the entire sequence. Bars, 10 µm.